ISCHEMIC INJURY AND PROTECTION Program Project Grant Departments of Physiology, Medicine & Anesthesiology David Geffen School of Medicine University of California, Los Angeles UCLA Principal Investigator/Program Director (Last, First, Middle): Ping, PROJECT 2: PKCe-DEPENDENT MODULATION OF VDAC AND ANT IN ISCHEMIC INJURY AND PROTECTION: IMPACT ON THE MPT PORE CLA Project Leader: Peipei Ping, Ph.D. Co-Project Leader: Ligia Toro, Ph.D. PHS 398 (Rev. 05/01) Page 121 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Ping, Project 2) Project 2 is designed to address the central theme of the PPG by examining kinase-dependent regulation of the mitochondrial permeability transition (MPT) pore in ischemic injury and protection. Project 2 complements the effort of Project 1 in characterizing signaling events that modulate mitochondrial permeability transition (MPT). Project 2 will delineate the molecular mechanism by which PKCe, a well-established cardioprotective kinase, interacts with and modifies key components of the MPT pore. The present studies are built upon our previous findings that the two core elements of the MPT pore, the outer membrane protein VDAC and the inner membrane protein ANT, are members of the PKCe sub-proteome at the mitochondria. These findings have been confirmed by the observation of strong localization of VDAC and ANT to PKCe immuno-complexes. Accordingly, Project 2 hypothesizes that PKCe interacts with, and modulates, VDAC and ANT via phosphorylation and that these post- translational modifications of VDAC and ANT impact their pore-forming abilities in the setting of ischemic injury and cardiac protection. Specifically, in collaboration with the Heart Biology Core, Project 2 will determine subcellular colocalization of PKCe with VDAC and ANT (using high resolution confocal imaging techniques) and will examine distribution of this kinase at the outer and inner mitochondrial membranes (by EM analyses). In collaboration with the Proteomic Core, Project 2 will determine the phosphorylation sites on endogenous VDAC and ANT in the in vivo setting. This knowledge in hand, Project 2 will take advantage of the strong expertise of the investigator team with membrane proteins to perform liposome reconstitution assays to conclusively examine the pore-forming abilities of wild type VDAC and ANT as well as their mutants either devoid of phosphorylation or constitutively activated. Furthermore, in collaboration with Project 4, Project 2 will determine how cell death pathway regulatory proteins (e.g., Bcl-2 and Bax) regulate the pore-forming abilities of VDAC and ANT and will examine whether the interactions of VDAC and ANT with these proteins are affected by PKCe-dependent phosphorylation. The proposed investigations will integrate information gained through membrane biophysics and proteomics studies with that from functional analysis at the organelle and system level. These studies will define, for the first time, the precise manner in which a cardioprotective kinase interacts with the MPT pore components; will map endogenous phosphorylation sites on VDAC and ANT in the normal myocardium and examine changes in these modifications that occur in the setting of cardioprotection; and will provide novel insights regarding how PKCe-VDAC-ANT complexes may be regulated by the Bcl-2 family of proteins.